SUMMARY PROJECT 3 The objective is to understand the energy driven cycles that lead to pumping substrates against the gradient of their concentration across membrane of the cell. Within the overall program whose goal is to define many states through the cycle, we seek to characterize states that are accessible to X-ray crystallography at atomic level. We will characterize those states using other techniques that determine the assembly of domains that move in the cycle, and critical intramolecular distances that feed into an integrative structure determination technology. We focus on one of these classes, termed ABC transporters that use ATP binding at two sites, and ATP hydrolysis as the energy source for transport of substrates. The two ATP binding cassettes (ABCs) interact with each other and relay structure change through scissor-like action to the substrate-binding site some 25 distant. We seek to define the structures of each of three ABC transporters, at multiple states in their pumping cycle. States are trapped using various nucleotide analogs that arrest the hydrolysis, and substrates arrested in transit. Any state that has or can be crystallized will be determined at atomic resolution by X-ray crystallography. Serial Femtosecond X-ray Free Electron Laser diffraction (SFX) will be harnessed to follow accessible steps in crystals initiated by laser-activated flash synchronized to X-ray pulses. The multiple states are characterized in solution by spectroscopies and X-ray scattering (SAXS) to define domain shifts. Mutations are used to introduce cysteines at specific sites that can form disufides to trap short-lived states in the process for structure analysis. Other pairs of cysteine substitutions are inserted for spin labeling. Spin labels are used for double electron-electron resonance (DEER) that serve to define the orientations of sites in the transmembrane domains and ABCs. The project seeks application to human ABC transporters subverted for multi-drug resistance, implicated in susceptibility to specific complex disorders, or used by viruses to cloak the immune response to them. Most structures of ABC transporters pertain to a single state. Through understanding the mechanisms of coupling energy to transport new targets for human therapeutic intervention will be uncovered.